The purpose of the proposed research is to characterize and to determine the significance of an endogenous phosphorylation (protein kinase and substrate) detected in the outer plasma membrane of normal and transformed mammalian cells. 3T3 cells incorporate 32P from gamma 32P ATP into phosphoserine and phosphothreonine residues of the surface proteins. This activity appears to be related to cell growth: it decreases as cell density increases; it is greater in growing than quiescent cells; and it is 5 to 10 fold greater in SV40 transformed 3T3 cells than in the normal 3T3. We plan to look more closely at the role of the protein kinase and substrate during stimulation of quiescent cells to enter G1 and S phase, and after viral transformation. In vitro and in vivo phosphorylation will be combined to determine the turnover and numbers of phosphorylated sites. SDS polyacrylamide gel electrophoresis and radioautography will be used to monitor incorporation into classes of proteins. The immediate and local effects of phosphorylation in the cell membrane will be studied with an electron spin labeling technique. We will also test the effects of phosphorylation on a variety of membrane functions, some of which are implicated in the stimulation of cells to grow, and some of which are reported to be influenced by ATP. BIBLIOGRAPHIC REFERENCES: Kinzel, V., Kubler, D., Mastro, A. M., and Rozengurt, E. Phosphoproteins of the cell surface as generated by endogenous or exogenous protein kinase: Stability of the 32P-labeled product. Biochim. Biophys. Acta 434:281-285 (1976). Mastro, Andrea and Rozengurt, E. Endogenous protein kinase in the outer plasma membrane of cultured 3T3 cells. J. Biol. Chem. 251:7849-7906 (1976).